Flow cytometry analysis of adhesion molecules on human Langerhans cells

Summary The Langerhans cell (LC) migrates between the epidermis and the regional lymph nodes to present antigens. This migration pattern requires the expression of a changing repertoire of cell‐surface molecules. In this work, we have investigated the expression of the adhesion molecules CD 11/CD 18 and CD 58 on LCs. Human epidermal cell suspensions were enriched in LCs (mean enrichment 75%) using a two‐step technique including a FicolI‐Hypaque gradient followed by Fc receptor panning with IgG‐coated sheep erythrocytcs. The number of cells obtained per experiment was 750000 (extremes 280000–1800000), and the following antibodies were tested on fresh suspensions and/or after 48 hours in culture: BB3 (antithyroglobulin negative control IgG2a), OKT6 (anti CDla, Ortho), anti HLA‐DR (Becton‐Dickinson), MHM 24 (anti CD 1la, leukocyte typing workshop n°3), MOl and 44 (anti CD l1b, leukocyte uping workshop n°3), anti CD l1c (Immunotech), 60‐3 and MHM 23 (anti CD 18, leukocyte typing workshop n°2), TS2/9‐1‐1 (anti CD 58, leukocyte typing workshop n°3). We found that amongst CD 11 subunits, only CD l1e was expressed in fresh suspensions, but was weaker than CD H, and disappeared w7ith culture. CD 58 was not detected in fresh suspensions but appeared after 2 days of culture, confirming earlier work. Thus the LC exhibits cell surface characteristics similar to tissue macrophages (CD 18 and CD 11c) prior to culture. The expression of CD 58 after culture is in accordance with the interaction of LC with CD2 hearing T‐lymphocytes during antigen presentation in peripheral lymph‐nodes. After homing to the epidermis, Langerhans ceils (LCs) originating from the hone marrow1 must interact in a variety of milieus to function efficiently as antigen‐presenting cells by trafficking mainly between the epidermis and regional lymph‐nodes.2 Adhesion molecules have been shown to be of the utmost importance in mediating further steps in cell activation.3 We first realized that adhesion was probably an important step in keratinocyte‐LC interactions when adding purified LCs suspensions to keratinocyte cultures and noted that LCs attached preferentially to keratinocytes as compared to blood mononuclear cells. Flow cytometry showed that the LFA‐1 β subumt (CD 18) and LFA‐3 (CD 58) were expressed by LCs after 48 h in culture with or without stimulation with lipopolysaccharide (LPS).4 Several investigators using various methods of purification or staining of LCs have since published comparabl